1. Field of the Invention
The present invention is broadly concerned with an improved high voltage capillary electrophoretic (CE) apparatus equipped with an easily constructed grounding joint intermediate to the ends of the capillary tube serving to electrically isolate an electrochemical detector from the high voltage applied potential. More particularly, it is concerned with such CE apparatus, and a method of fabricating the ground joint tube assembly thereof, wherein a capillary tube is scored, a flexible, polymeric, ion-permeable sleeve is applied over the score and sealed, and the tube is fractured. The resultant tube assembly gives excellent, reproducible CE results.
2. Description of the Prior Art
Since its introduction over a decade ago, capillary electrophoresis has established itself as a powerful analytical tool for the separation and analysis of complex mixtures (Jorgenson et al., Anal. Chem., 53 (1981) 1298). In CE systems, a fused silica capillary is filled with buffer and a large voltage is applied. By virtue of the presence of ionized silanol groups on the capillary wall, the applied voltage creates electroosmotic flow which allows the charge-independent detection of species injected onto the column. CE capillaries with small diameters is advantageous over conventional slab gel electrophoresis due to higher efficiency, lower joule heating effect and faster analysis times.
One of the main areas of present-day CE research is the development of sensitive detection systems. Because of the small injection volumes involved in CE, high sensitivity/small volume detectors are necessary. UV detection is the most commonly employed detection method for CE. However, since this is an optical technique and is pathlength dependent, the sensitivity is limited when using small diameter capillaries. Laser-based fluorescence detectors are more sensitive, but are expensive and limited to certain wavelengths. Electrochemical detection has an advantage over these methods in that the response is not dependent on pathlength; therefore, very small capillary diameters can be used without sacrifice in signal. It also utilizes relatively inexpensive instrumentation. However, electrochemical detection presents a serious difficulty in that the detection apparatus must be shielded from the high voltage used in the CE separation. If this is not done, the sensitive detection apparatus may be damaged, and very low signal-to-noise ratios will be obtained.
Wallingford and Ewing (Anal. Chem., 53 (1987) 1762; Anal. Chem., 61 (1989) 98) have developed an off-column electrochemical detector, and have reported 10.sup.-8 M detection limits for several catechol compounds. In their system, two pieces of capillary column are coupled inside a piece of rigid porous glass capillary. This joint permits the flow of ions but not bulk electrolytic flow, enabling the detection end of the capillary to be held at ground. The fabrication of this joint assembly is quite difficult and intricate. Unless perfect alignment of both sections of capillary is achieved, considerable band broadening can occur. The joint does not appear to be durable as the porous glass is extremely fragile and must be kept submerged in solution. Another limitation of this design is that the porous glass capillary is not readily available. Huang and Zare (Anal. Chem., 62 (1990) 443) designed an on-column frit which also served to isolate the final section of the capillary column from the applied electrical field. However, the authors describe the drawbacks of this design, including lack of capillary-to-capillary reproducibility, leakage of the frit and difficulty of fabrication (requiring the use of a CO.sub.2 laser). Recently, Huang et al. (Anal. Chem., 63 (1991) 189) have reported that it is not necessary to isolate the microelectrode from the high voltage if capillaries with very small internal diameter (5 .mu.m) are employed. In such small capillaries, the current generated by the CE separation is low enough that it does not adversely affect the electrochemical detection. However, as a consequence of the small size, the concentration detection limits are not as low as those reported with larger internal diameter capillary columns.